EP2205973B1 - Procédé et dispositif de bioanalyse multiplexée à base d'une nanotechnologie de grande sensibilité - Google Patents

Procédé et dispositif de bioanalyse multiplexée à base d'une nanotechnologie de grande sensibilité Download PDF

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EP2205973B1
EP2205973B1 EP08833536A EP08833536A EP2205973B1 EP 2205973 B1 EP2205973 B1 EP 2205973B1 EP 08833536 A EP08833536 A EP 08833536A EP 08833536 A EP08833536 A EP 08833536A EP 2205973 B1 EP2205973 B1 EP 2205973B1
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analyte
nanoparticles
probes
type
complementary
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EP2205973A2 (fr
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Gianfranco Liguri
Francesco Trisolini
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HOSPITEX DIAGNOSTICS S.R.L.
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Hospitex Diagnostics Srl
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • This invention refers to a method and a device for the high sensitivity analysis of multiple analytes in biological samples.
  • the invention refers to a method and a device for the detection of complementary pairs of molecules, such as nucleic acid-nucleic acid (e.g. DNA-DNA, DNA-RNA, RNA-RNA) or protein-protein (e.g. antigen-antibody) pairs.
  • nucleic acid-nucleic acid e.g. DNA-DNA, DNA-RNA, RNA-RNA
  • protein-protein e.g. antigen-antibody
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • Quantitative PCR which is based on the amplification of target DNA, is another common modification of PCR, but is only able to generate semi-quantitative data (number of copies of a DNA sequence in a sample) and can be affected by the presence of inhibitors and low copy number templates.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • biochemistry is used to detect the presence of an antigen that is characteristic of a particular pathogenic organism, or to measure the concentration of antibodies in blood plasma (as in AIDS tests, for example).
  • This technique relies on specific antigen-antibody reactions that are made visible by various procedures.
  • many other ELISA-related techniques have been developed. One of them is the "Two-hybrid capture” technique, developed by Digene Corp. for the detection of human papilloma virus (HPV), which is able to detect RNA:DNA hybrids using an amplified chemiluminescent signal.
  • HPV human papilloma virus
  • a DNA microarray is a collection of microscopic DNA probes arrayed on a solid surface, such as glass or plastic, that bind to chemically suitable, complementary targets that have been previously amplified and tagged with a fluorescent molecule, which permits detection of fluorescent signals generated upon hybridization.
  • These techniques therefore permit multiplexing, but require prior target DNA amplification to ensure optimal sensitivity, as in the case of RT-PCR-amplified mRNA detection. As such, they are affected by the aforementioned drawbacks of PCR.
  • Microarrays also require very expensive scanners and sophisticated detection systems, which consist of specific focalized lasers that reveal every single micro-spot. Moreover, they are not easily automated and are therefore not very useful in diagnostics. In addition to this, they are also affected by the aforementioned limitations of fluorescent detection.
  • a conductivity-based DNA detection method utilizing oligonucleotide-functionalized Au nanoparticles is described by Park et al. in "Array-Based Electrical Detection of DNA with Nanoparticles Probes", Science, vol. 295, pages 1503-1506 .
  • W003/095973A2 discloses an analyte detection method based on the use of Raman labelled nanoparticles that can be activate to provide SERS effect.
  • a DNA detection method based on latex microsphere and gold nanoparticles is described by Reynolds III et al. in "A gold nanoparticle/latex microsphere-based colorimetric oligonucleotide detection method", Pure Appl. Chem., Vol. 72, Nos. 1-2, pp. 229-235, 2000 .
  • Gold and latex probes are linked together by the target DNA strand, generating a white-to red color change in a size-selective membrane through which excess or nonhybridized gold particles are separated from latex indicator probes.
  • the aim of this invention is to deliver an analytical biotechnology technique that is able to carry out detection of complementary pairs of molecules, such as nucleic acid-nucleic acid (DNA-DNA, DNA-RNA, RNA-RNA) or protein-protein pairs, without the drawbacks of other techniques.
  • complementary pairs of molecules such as nucleic acid-nucleic acid (DNA-DNA, DNA-RNA, RNA-RNA) or protein-protein pairs
  • the object of the invention is to provide a method and a device for the simultaneous and quantitative detection of different subtypes (multiplexing) of a given biological target system (virus, genetic mutations, etc.).
  • the aforementioned aims are achieved using a method and a device that exploits the interaction of non-biological nanoparticles with biological structures (such as nucleic acids and proteins) to determine dimensional increases, thereby permitting the detection of a biological target (virus, genetic mutations, etc.).
  • biological structures such as nucleic acids and proteins
  • the proposed method and device make use of a microarray architecture of nanoparticles linked to molecular probes or specific antibodies, which are adsorbed onto a solid surface and preferably arranged in a monolayer.
  • the aim of the system is to specifically detect complementary pairs of molecules, such as protein-protein pairs (e.g. antigen-antibody complexes) and DNA-DNA or DNA-RNA pairs (e.g. probe-target complexes).
  • protein-protein pairs e.g. antigen-antibody complexes
  • DNA-DNA or DNA-RNA pairs e.g. probe-target complexes
  • focalized laser sources coupled to a system of photosensors can preferably be used.
  • Various detection strategies can then be exploited, such as image analysis or light scattering.
  • binding to two other components Y,Z of the complementary element of the other pair is determined (in the illustrated example, two elements complementary to two different terminal portions of the target), one attached to a nanoparticle N and the other to a solid support S.
  • latex nanoparticles N that are monodispersed in the suspension under test, specifically bind to the target X through complementary molecular probes Y that are attached to the nanoparticles.
  • These nanoparticle-probe-target complexes diffuse by Brownian motion from the liquid suspension to the solid-liquid interface and dock onto the solid surface S (e.g. one of the internal surfaces of the tube containing the suspension) as a monolayer, resulting in the target X binding to other specific molecular probes Z (which are complementary to other portions of the target), that are also adsorbed onto the solid surface.
  • the proposed method guarantees rigidity and reliability, as well as rapid analysis and automation. Moreover, less time and money will be required to train personnel, compared to previous techniques.
  • the method and the device according to the present invention enable two crucial aims in the medical diagnostic field to be fulfilled.
  • the first of these is signal enhancement, in addition to better use of the solid space and selective concentration of the molecule to be analysed in a two-dimensional state, and secondly, the ability to carry out more simultaneous tests, in the same reactor and on the same sample (multiplexing), resulting in a considerable saving of time, money and biological material.
  • this last feature is especially advantageous when it comes to detecting genetic mutations in human samples and can also be used to genotype viruses that are dangerous to humans.
  • the genome (G1-G4) of the various HPV subtypes is the target to be detected.
  • the probes (C) attached to the nanoparticles (N) are DNA probes that are complementary to the conserved regions of the HPV genome (and are therefore able to detect any HPV subtype), whereas the probes (V1-V4) attached to the solid support (S) are DNA probes that are complementary to the variable regions of the HPV genome (therefore, they can specifically detect a particular HPV subtype). As a consequence, this permits the spatial detection of various subtypes (multiplexing).
  • the system foresees the use of a plastic container (e.g. a tube), on one of whose internal surfaces (S) an area of approximately 1 cm 2 is selected and sub-divided into different cells (1-4).
  • a plastic container e.g. a tube
  • S internal surfaces
  • V1-V4 oligonucleotide
  • V1-V4 corresponding to a particular DNA probe that is complementary to the variable part of the genome of a specific HPV genotype
  • the biological sample is added to the tube so that qualitative and quantitative measurements of HPV can be made.
  • a specific target e.g. G1
  • binding occurs between the target and a nanoparticle (N), through its conserved DNA portion (C), and with portion (I) of the internal surface (S) of the tube, through its variable DNA portion (V1), resulting in docking of the nanoparticles onto the internal surface of the tube ( Fig. 3 ).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Nanotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)
  • Measurement Of Radiation (AREA)

Claims (10)

  1. Une méthode de titrage biologique multiple haute sensibilité, caractérisée par le fait qu'elle comprend les étapes suivantes :
    - la fourniture d'une série de nanoparticules de latex ou de silice ;
    - l'application d'au moins une sonde d'un premier type ayant une affinité avec, au moins, un analyte de chaque nanoparticule ;
    - l'application d'une série de sondes, d'au moins d'un seconde type ayant une affinité avec, au moins, ledit analyte, sur la surface d'un support permettant la détection dudit analyte dans l'espace ;
    - le mélange de ladite série de nanoparticules avec un échantillon liquide déterminant ainsi le jumelage d'une ou plusieurs desdites nanoparticules, si contenues dans l'échantillon, comme conséquence du couplage entre la sonde du premier type et l'analyte ;
    - la mise en contact desdites nanoparticules liées à l'analyte avec ledit support afin de provoquer l'attachement à la surface, comme conséquence du couplage entre les sondes du second type et ledit analyte ;
    - la détection dudit analyte par l'analyse du signal lumineux généré par les nanoparticules liées à la surface dès lors qu'elles sont illuminées.
  2. Une méthode suivant la revendication 1 où lesdites sondes du premier type sont complémentaires à une zone conservée dudit analyte et lesdites sondes du second type sont complémentaires aux zones variables dudit analyte.
  3. Une méthode suivant la revendication 2 où ledit support permet la détection dans l'espace des différents sous-types dudit analyte.
  4. Une méthode suivant la revendication 3 où la surface dudit support est divisée en plusieurs zones et, au moins, une sonde qui est complémentaire à l'une des zones variables dudit analyte, est appliquée à chacune des zones, donnant ainsi comme résultat la fixation des nanoparticules liées aux différents sous-types d'analyte dans les différentes zones.
  5. Une méthode suivant l'une des revendications précédentes où le signal lumineux généré par les nanoparticules liées à la surface est analysé par le biais de techniques de diffusion de la lumière, d'analyse d'image et d'ondes évanescentes.
  6. Une méthode suivant l'une des revendications précédentes où lesdites nanoparticules ont une taille de 100 - 300 mm.
  7. Un dispositif de titrage biologique multiplexe haute sensibilité adapté à la méthode citée dans la revendication 1, caractérisé par le fait qu'il comprend :
    - un réceptacle dans lequel l'échantillon liquide peut être analysé :
    - une série de nanoparticules de latex ou de silice dispersées dans le liquide, et dont chaque nanoparticule est attachée à, au moins, une sonde d'un premier type ayant une affinité avec, au moins, un analyte ;
    - une série de sondes, au moins, du second type ayant une affinité avec ledit analyte et étant attachées à la surface d'au moins une paroi du réceptacle ;
    - une source laser concentrée pour l'illumination des nanoparticules présentes sur la surface de la paroi, résultant des fixations ayant eu lieu entre les sondes liées aux nanoparticules et l'analyte et entre l'analyte et les sondes attachées à la surface ;
    - des moyens photosenseurs pour la détection du signal lumineux généré par les nanoparticules liées à la surface dès lors qu'elles sont illuminées.
  8. Un dispositif suivant la revendication 7 où le signal lumineux généré par les nanoparticules liées à la surface est analysé par le biais de techniques de diffusion de la lumière, d'analyse d'image et d'ondes évanescentes.
  9. Un dispositif suivant la revendication 7 ou 8 où la surface de ladite paroi est divisée en plusieurs zones, avec, au moins, chaque zone liée à, au moins, une sonde qui est complémentaire à l'une des différentes zones variables dudit analyte, donnant ainsi la fixation des nanoparticules liées aux différents sous-types d'analyte dans les différentes zones.
  10. Une méthode et un dispositif suivant l'une des revendications précédentes pour la détection des différents génotypes du virus du papillome humain (HPV).
EP08833536A 2007-09-26 2008-09-26 Procédé et dispositif de bioanalyse multiplexée à base d'une nanotechnologie de grande sensibilité Not-in-force EP2205973B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000023A ITPO20070023A1 (it) 2007-09-26 2007-09-26 Metodo e dispositivo per l'analisi multipla ad alta sensibilita di campioni biologici
PCT/IB2008/002526 WO2009040652A2 (fr) 2007-09-26 2008-09-26 Procédé et dispositif de bioanalyse multiplexée à base d'une nanotechnologie de grande sensibilité

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EP2205973A2 EP2205973A2 (fr) 2010-07-14
EP2205973B1 true EP2205973B1 (fr) 2013-03-06

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US (1) US20100240147A1 (fr)
EP (1) EP2205973B1 (fr)
ES (1) ES2411064T3 (fr)
IT (1) ITPO20070023A1 (fr)
WO (1) WO2009040652A2 (fr)

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EP2524219A4 (fr) 2010-01-14 2013-08-07 Univ Central Florida Res Found Procédés de détection et d'analyse de biomolécules et de complexes de biomolécules (bmc) et leur utilisation pour la recherche et le diagnostic médical

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WO2002044725A1 (fr) * 2000-11-30 2002-06-06 Innotrac Diagnostics Oy Dosage bioanalytique

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DE69010089T2 (de) 1989-01-20 1994-12-22 Stago Diagnostica Submikronische teilchen, herstellung und verwendung in der immundiagnose.
TW239881B (fr) * 1992-12-22 1995-02-01 Sienna Biotech Inc
US6200820B1 (en) * 1992-12-22 2001-03-13 Sienna Biotech, Inc. Light scatter-based immunoassay
US7169556B2 (en) * 1996-07-29 2007-01-30 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
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US20030211488A1 (en) * 2002-05-07 2003-11-13 Northwestern University Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection
US7122384B2 (en) * 2002-11-06 2006-10-17 E. I. Du Pont De Nemours And Company Resonant light scattering microparticle methods

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WO2002044725A1 (fr) * 2000-11-30 2002-06-06 Innotrac Diagnostics Oy Dosage bioanalytique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REYNOLDS III, R.A., MIRKIN, C.A., LETSINGER, R.: "A gold nanoparticle/latex microsphere-based colorimetric oligonucleotide detection method", PURE APPL CHEM, vol. 72, no. 1-2, 2000, pages 229 - 235 *
ZHAO XIAOJUN ET AL: "Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 125, no. 38, 24 September 2003 (2003-09-24), pages 11474 - 11475, ISSN: 0002-7863 *

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EP2205973A2 (fr) 2010-07-14
WO2009040652A3 (fr) 2010-04-29
WO2009040652A2 (fr) 2009-04-02
ES2411064T3 (es) 2013-07-04
US20100240147A1 (en) 2010-09-23
ITPO20070023A1 (it) 2009-03-27

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